LOCUS       sequence-178688-        5224 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial co-expression of folA (DHFR) and thyA(TS) with a barcode 
            within the noncoding region between the genes .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5224)
  AUTHORS   Schober AF, Mathis AD, Ingle C, Park JO, Chen L, Rabinowitz JD, 
            Junier I, Rivoire O, Reynolds KA
  TITLE     A Two-Enzyme Adaptive Unit within Bacterial Folate Metabolism.
  JOURNAL   Cell Rep. 2019 Jun 11;27(11):3359-3370.e7. doi: 
            10.1016/j.celrep.2019.05.030.
  PUBMED    31189117
REFERENCE   2  (bases 1 to 5224)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.celrep.2019.05"; journalName: "Cell Rep"; date: 
            "2019-06-11- 11"; volume: "27"; issue: "11"; pages: "3359-3370.e7"
COMMENT     Sequence Label: pACYCDuet_D27NDHFR_WTTS
FEATURES             Location/Qualifiers
     source          1..5224
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        77..95
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    96..120
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             982..1026
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1064..1082)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1078..1125
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1346..2005)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1920..1939
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2006..2108)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(2500..2517)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2634..3179)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(2714..2733)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3404..4486)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4448..4467
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4487..4564)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        4610..4628
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4629..4653
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             4709..4726
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4739..5218
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDRVIGMENAMPWNLPANLAWFKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
ORIGIN
        1 tcgacaagct tgcggccgca taatgcttaa gtcgaacaga aagtaatcgt attgtacacg
       61 gccgcataat cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
      121 ccatcttagt atattagtta agtataagaa ggagatatac atatggcaga tctcatgaaa
      181 cagtacctgg agctgatgca aaaagttctg gatgagggta cccagaaaaa cgaccgcacg
      241 ggtaccggaa cgctgagcat ttttggccat cagatgcgtt ttaacctgca ggacggattc
      301 ccgctggtta cgaccaaacg ctgccacctg cgtagcatta ttcatgagct gctgtggttt
      361 ctgcaaggtg acactaacat cgcgtatctg cacgaaaaca atgtgacgat ctgggatgaa
      421 tgggccgatg aaaacggcga tctgggccca gtgtatggta aacagtggcg cgcctggcca
      481 acgccagatg gccgccacat cgatcagatc acgaccgtgc tgaatcaact gaaaaacgac
      541 ccggacagcc gccgcattat tgtttccgcg tggaatgtgg gtgaactgga taaaatggcg
      601 ctggcgccgt gccatgcatt cttccagttt tacgttgcgg acggtaagct gagctgtcaa
      661 ctttatcagc gcagctgcga tgttttcctc ggcctgccgt tcaatatcgc cagctacgcg
      721 ttactggtgc atatgatggc gcagcagtgc gacctggagg tgggtgattt tgtctggacc
      781 ggcggtgata cccacctgta cagcaaccat atggaccaaa cgcatctgca attgagccgt
      841 gagccgcgtc cgctgccgaa actgattatt aagcgcaaac ccgagagcat ctttgactat
      901 cgtttcgaag acttcgagat tgaaggctac gatccgcacc cgggtatcaa agcgccggtg
      961 gctatctaac tcgagtctgg taaagaaacc gctgctgcga aatttgaacg ccagcacatg
     1021 gactcgtcta ctagcgcagc ttaattaacc taggctgctg ccaccgctga gcaataacta
     1081 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa acctcaggca
     1141 tttgagaagc acacggtcac actgcttccg gtagtcaata aaccggtaaa ccagcaatag
     1201 acataagcgg ctatttaacg accctgccct gaaccgacga ccgggtcgaa tttgctttcg
     1261 aatttctgcc attcatccgc ttattatcac ttattcaggc gtagcaacca ggcgtttaag
     1321 ggcaccaata actgccttaa aaaaattacg ccccgccctg ccactcatcg cagtactgtt
     1381 gtaattcatt aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa
     1441 tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccata gtgaaaacgg
     1501 gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg
     1561 gattggctga gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt
     1621 caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt
     1681 attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt
     1741 gaacactatc ccatatcacc agctcaccgt ctttcattgc catacggaac tccggatgag
     1801 cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct
     1861 ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag
     1921 caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg
     1981 tatatccagt gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact
     2041 caaaaaatac gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt
     2101 gccgatcaac gtctcatttt cgccaaaagt tggcccaggg cttcccggta tcaacaggga
     2161 caccaggatt tatttattct gcgaagtgat cttccgtcac aggtatttat tcggcgcaaa
     2221 gtgcgtcggg tgatgctgcc aacttactga tttagtgtat gatggtgttt ttgaggtgct
     2281 ccagtggctt ctgtttctat cagctgtccc tcctgttcag ctactgacgg ggtggtgcgt
     2341 aacggcaaaa gcaccgccgg acatcagcgc tagcggagtg tatactggct tactatgttg
     2401 gcactgatga gggtgtcagt gaagtgcttc atgtggcagg agaaaaaagg ctgcaccggt
     2461 gcgtcagcag aatatgtgat acaggatata ttccgcttcc tcgctcactg actcgctacg
     2521 ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa cggggcggag atttcctgga
     2581 agatgccagg aagatactta acagggaagt gagagggccg cggcaaagcc gtttttccat
     2641 aggctccgcc cccctgacaa gcatcacgaa atctgacgct caaatcagtg gtggcgaaac
     2701 ccgacaggac tataaagata ccaggcgttt ccccctggcg gctccctcgt gcgctctcct
     2761 gttcctgcct ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt gtctcattcc
     2821 acgcctgaca ctcagttccg ggtaggcagt tcgctccaag ctggactgta tgcacgaacc
     2881 ccccgttcag tccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccgga
     2941 aagacatgca aaagcaccac tggcagcagc cactggtaat tgatttagag gagttagtct
     3001 tgaagtcatg cgccggttaa ggctaaactg aaaggacaag ttttggtgac tgcgctcctc
     3061 caagccagtt acctcggttc aaagagttgg tagctcagag aaccttcgaa aaaccgccct
     3121 gcaaggcggt tttttcgttt tcagagcaag agattacgcg cagaccaaaa cgatctcaag
     3181 aagatcatct tattaatcag ataaaatatt tctagatttc agtgcaattt atctcttcaa
     3241 atgtagcacc tgaagtcagc cccatacgat ataagttgta attctcatgt tagtcatgcc
     3301 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     3361 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     3421 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     3481 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     3541 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     3601 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     3661 tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg
     3721 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     3781 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     3841 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     3901 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     3961 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     4021 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     4081 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     4141 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     4201 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     4261 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     4321 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     4381 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     4441 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     4501 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     4561 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaaatt aatacgactc
     4621 actatagggg aattgtgagc ggataacaat tcccctgtag aaataatttt gtttaacttt
     4681 aataaggaga tataccatgg gcagcagcca tcaccatcat caccacagcc aggatccgat
     4741 gatcagtctg attgcggcgt tagcggtaga tcgcgttatc ggcatggaaa acgccatgcc
     4801 gtggaacctg cctgccaacc tcgcctggtt taaacgcaac accttaaata aacccgtgat
     4861 tatgggccgc catacctggg aatcaatcgg tcgtccgttg ccaggacgca aaaatattat
     4921 cctcagcagt caaccgggta cggacgatcg cgtaacgtgg gtgaagtcgg tggatgaagc
     4981 catcgcggcg tgtggtgacg taccagaaat catggtgatt ggcggcggtc gcgtttatga
     5041 acagttcttg ccaaaagcgc aaaaactgta tctgacgcat atcgacgcag aagtggaagg
     5101 cgacacccat ttcccggatt acgagccgga tgactgggaa tcggtattca gcgaattcca
     5161 cgatgctgat gcgcagaact ctcacagcta ttgctttgag attctggagc ggcggtaacc
     5221 actg
//
